Von Willebrand factor (VWF) is a high molecular weight, multimeric glycoprotein in blood plasma, which has important functions in the process of primary hemostasis. VWF possesses, inter alia, binding sites for collagen and for glycoprotein Ib (GPIb) which is located on the surface of platelets. GPIb is an integral membrane protein which, together with another integral membrane protein, glycoprotein IX (GPIX), forms the glycoprotein Ib-IX-receptor complex in the platelet membrane. GPIb is a two-chain molecule comprising a heavy chain with an apparent molecular mass of about 145 kDa (synonym: alpha chain or GPIb.alpha.) and a light chain with an apparent molecular mass of about 22 kDa (synonym: beta chain or GPIb.beta.), which are connected to one another by disulfide bonds (Lopez, J. A. et al., Cloning of the .alpha. chain of human platelet glycoprotein Ib: A transmembrane protein with homology to leucine-rich .alpha..sub.2-glycoprotein. Proc. Natl. Acad. Sci. USA 1987, 84: 5615-5619). Glycocalicin is a fragment of the GPIb.alpha. chain, which is proteolytically removed from the intact receptor in the platelet membrane. Glycocalicin is detectable in plasma. Increased plasma concentrations of free glycocalicin indicate a disorder of platelet function (Beer, J. H. et al., Glycocalicin: A New Assay—The Normal Plasma Levels And Its Potential Usefulness in Selected Diseases. Blood 1994, 83(3): 691-702).
In the case of vascular injury, collagen surfaces are exposed to which VWF binds. Due to its binding to collagen and under the influence of increased shear forces acting on the collagen-bound VWF, VWF is altered or activated in such a way that it can bind to the amino-terminal end of the GPIb heavy chain (GPIb.alpha.) in the GPIb-IX-receptor complex of the platelet membrane. In this way, the activated VWF captures passing platelets, resulting in the formation of a first agglomerate of VWF, collagen and platelets at the site of the injury. Subsequently, the platelets are activated, thereby also starting plasmatic coagulation which finally, after multiple amplifying cascades and attachment of further platelets, results in the wound being closed. VWF-GPIb interaction disorders increase hemorrhagic tendencies.
Qualitative or quantitative VWF disorders cause what is known as a von Willebrand syndrome (synonym: von Willebrand disease, VWD), one of the most common inheritable hemorrhagic conditions. Various screening processes are available for diagnosing a von Willebrand syndrome, for example bleeding time (BT) determination, quantitative processes for determining the concentration of VWF antigen (VWF:Ag), such as ELISA assays for example, and processes for determining VWF activity, such as ristocetin-induced platelet agglutination (VWF:RCo).
The newest generation of functional assays for determining VWF activity involves determining the ability of VWF to bind GPIb.alpha.
Assays have been disclosed which comprise using wild-type GPIb.alpha. and determining VWF binding to GPIb.alpha. in the presence of ristocetin (WO 01/02853 A2; Vanhoorelbeke, K. et al., A reliable von Willebrand factor: Ristocetin cofactor enzyme-linked immunosorbent assay to differentiate between type 1 and type 2 von Willebrand disease. Semin Thromb Hemost. 2002, 28(2): 161-165; Federici, A. B. et al., A sensitive ristocetin co-factor activity assay with recombinant glycoprotein Ib.alpha. for the diagnosis of patients with low von Willebrand factor levels. Haematologica 2004, 89(1): 77-85).
Other assays have been disclosed which make use of GPIb.alpha. “gain-of-function” mutations which are known to have a higher affinity for VWF and interact with VWF more strongly than wild-type GPIb.alpha. protein. These assays enable VWF binding to the mutated GPIb.alpha. to be determined in the absence of ristocetin (WO 2009/007051 A2 or WO 2009/026551 A1).
Defects of the GPIb protein likewise cause hemorrhagic conditions. Gain-of-function mutations of the GPIb protein are the cause of platelet-type von Willebrand syndrome (PT-VWD), an autosomally dominantly transmitted hemorrhagic condition. Substitution of the methionine residue in position 239 of the GPIb.alpha. chain by a valine residue (M239V) has been described by Russell & Roth (Russell, S. D. & Roth, G. J., Pseudo-von Willebrand Disease: A mutation in the platelet glycoprotein Ib.alpha. gene associated with a hyperactive surface receptor. Blood 1993, 81(7): 1787-1791). Mutations in position 233 of the GPIb.alpha. chain may also cause PT-VWD (Matsubara, Y. et al., Identification of a novel point mutation in platelet glycoprotein Ib.alpha., Gly to Ser at residue 233, in a Japanese family with platelet-type von Willebrand disease. Journal of Thrombosis and Haemostasis 2003, 1: 2198-2205).
If a patient is diagnosed with an increased tendency toward hemorrhage, the cause of the disorder must be detected in order to be able to initiate a suitable therapy. Owing to the multiplicity of possible disorders which may cause an increased tendency toward hemorrhage, the availability of screening assays that firstly enable the functionality of certain parts of the coagulation system to be investigated is desirable in clinical diagnostics. If a disorder can be located in a certain part with the aid of such a screening assay, specific individual assays may be carried out in order to specify the exact cause. If no disorder is found in a certain part with the aid of a screening assay, specific individual assays need not be carried out.
The object addressed by the present invention was therefore that of providing a screening process which enables VWF-GPIb interaction disorders to be detected. Such a process should be equally sensitive to VWF disorders and GPIb protein disorders.
VWF-GPIb interaction disorders may be caused, for example, by
a) quantitative or qualitative disorders of the VWF protein, such as, for example, states of abnormal deficiency, absence of the large multimers, lack of factor VIII binding ability;
b) VWF inhibitors such as, for example, autoantibodies against VWF which prevent VWF from binding to GPIb, or increased plasma concentrations of glycocalicin which occupies the VWF binding sites and thus diminishes VWF activity, or VWF-inhibiting therapeutic drugs such as, for example, ARC1779, a VWF-binding aptamer, or AJW200, a humanized monoclonal anti-VWF antibody (Firbas, C. et al., Targeting von Willebrand factor and platelet glycoprotein Ib receptor. Expert Rev. Cardiovasc. Ther. 2010, 8 (12): 1689-1701), or GPIb fragment employed as a therapeutic drug (Hennan, J. K. et al., Pharmacologic inhibition of platelet vWF-GPIb.alpha. interaction prevents coronary artery thrombosis. Thromb Haemost 2006, 95: 469-75);
c) VWF activators;
d) qualitative disorders of the GPIb protein, such as, for example, gain-of-function mutations having a higher affinity for VWF and therefore accelerating VWF breakdown;
e) GPIb inhibitors such as, for example, autoantibodies against GPIb which prevent VWF from binding to GPIb, or therapeutic drugs such as H6B4-Fab for example, the Fab fragment of a humanized monoclonal anti-GPIb.alpha. antibody (see likewise Firbas, C. et al.);
f) GPIb activators (e.g. thrombin).
The object is achieved by contacting the sample from a patient with isolated GPIb.alpha. protein, with VWF protein and with a solid phase associated with a GPIb.alpha.-specific antibody, and determining formation of a complex between VWF protein, GPIb.alpha. protein and the solid phase. VWF-GPIb interaction is disordered if said complex formation is reduced or increased compared to normal. Such samples should then be tested specifically for VWF and GPIb disorders with the aid of specific individual assays.